Multiple antibiotic resistance operon assays

ABSTRACT

An isolated and cloned region of a bacterial chromosome containing a multiple antibiotic resistance operon is disclosed. A description of the structure and function of the operon is provided as are assorted recombinant DNA constructs involving the operon or fragments thereof. The diagnostic, therapeutic and experimental uses of these constructs are also disclosed. Methods of evaluating the antibiotic effectiveness of compositions are disclosed and methods of treatment employing effective compositions are provided.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.07/938,085 filed Aug. 28 1992, abandoned and entitled "MultipleAntibiotic Resistance Regulon Assays," the entire disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of antimicrobial therapy.In particular, the present invention relates to methods and productsuseful in inhibiting the growth of bacteria or other microbes. Inaddition, this invention relates to identifying loci in bacteria orother microbes which affect antibiotic or antimicrobial susceptibilityand to the production of bacterial strains useful in the field ofantimicrobial therapy.

BACKGROUND OF THE INVENTION

Antibiotic or antimicrobial substances have long been used to inhibitthe growth of bacteria or other microbes and to treat bacterial ormicrobial infections in humans, other animals, and in tissue culture.The use of antibiotics or antimicrobials in a treatment regimen,however, has the undesirable effect of selecting for bacteria or othermicrobes which are resistant to those antibiotics or antimicrobialswhich are administered or applied. As a result, treatment regimens canbe adversely affected or, in some cases, rendered ineffective. Thisnecessitates a continual search for new antibiotics and antimicrobials.

Of particular interest is the discovery of bacteria which express amultiple antibiotic resistance phenotype (Mar). This phenotype entailssimultaneous resistance to a multiplicity of antibiotics which areunrelated in chemical structure. The appearance of such bacteria andinfections by such bacteria greatly increase the difficulty ofidentifying effective antibiotics and treating infections in humans orother animals.

Multiple antibiotic resistance in bacteria is most commonly associatedwith the presence of plasmids which contain one or more resistancegenes, each encoding a single antibiotic resistance phenotype (Clewell1981; Foster 1983). Multiple antibiotic resistance associated with thechromosome, however, has been reported in Klebsiella, Enterobacter,Serratia (Gutmann et al. 1985), Neisseria (Johnson and Morse 1988), andEscherichia (George and Levy 1983a).

Bacteria expressing the multiple antibiotic resistance phenotype can beisolated by selecting bacteria with a single antibiotic and thenscreening for cross-resistance to structurally unrelated antibiotics.For example, George and Levy initially described a chromosomal multipleantibiotic resistance system which exists in Escherichia coli and whichcan be selected by a single drug, e.g., tetracycline or chloramphenicol(George and Levy 1983a). In addition to resistance to the selectiveagents, the Mar phenotype includes resistance to structurally unrelatedagents, including nalidixic acid, rifampin, penicillins, andcephalosporins (George and Levy 1983); more recently, resistance to thefluoroquinolones has been described (Cohen et al. 1989).

The expression of a Mar phenotype, conferring substantially increased,simultaneous and coordinated resistance to a multiplicity ofstructurally unrelated compounds, appears to involve coordinated changesin the expression of a multiplicity of loci. This has been demonstratedin Mar phenotype bacteria of the species E. coli (Cohen et al. 1989).Such coordinated control of the expression of a multiplicity of lociimplies the existence of an operon which directly or indirectlyregulates the expression of the multiplicity of loci directlyresponsible for the Mar phenotype. One locus in one such operon wasidentified in E. coli and named marA by George and Levy (George and Levy1983b).

Prior to the present invention, however, no multiple antibioticresistance (mar) operon had been isolated or cloned. In addition, no maroperon had been characterized as to its structure and operation so as toenable the use of such an operon or its fragments for diagnostic,therapeutic or experimental purposes. Finally, the several othercontributions to the field of antibacteriology in the claims wereunavailable to those skilled in the art prior to the present invention.

SUMMARY OF THE INVENTION

The present invention relates generally to developing and evaluatingantibiotic treatments effective against bacteria possessing a multipleantibiotic resistance (mar) operon. Because the expression of such anoperon causes bacteria to become simultaneously resistant to amultiplicity of structurally unrelated antibiotics, it is a generalobject of the present invention to provide methods and compositionsuseful in combating bacteria possessing a mar operon or exhibiting a Marphenotype. It is one particular object of the present invention toprovide tests for compositions which are effective against bacteriaexpressing a Mar phenotype but which do not induce the expression of amar operon, or which inhibit the expression of a mar operon. To thisend, it is also an object of the present invention to provide clonednucleotide sequences, as well as bacterial cells expressing suchsequences, which are useful in performing such tests and ininvestigating bacterial multiple antibiotic resistance operons.

The present invention provides cloned bacterial mar operons and clonedfragments thereof. In particular, a cloned repressor locus and a clonedactivator locus of a mar operon, as well as cloned loci encodinganti-sense transcripts to the repressor and activator loci, areprovided. Using such clones, substantially pure repressor protein andsubstantially pure activator protein are provided. In addition, usingsuch clones, isolated nucleotide sequences, either sense or anti-senseto those loci, are provided. These sequences are useful as probes forsubstantially homologous loci in other species including bacteria,fungi, parasites, and animal cells and are useful for altering theexpression of a Mar phenotype in bacteria, either by encoding repressoror activator proteins or by encoding anti-sense transcripts whichinhibit the expression of either a mar repressor or mar activator locus.

The present invention also provides cloned nucleotide sequences in whichthe regulatory region of a mar operon has been operably joined to amarker locus. Such sequences are useful in assaying the effect ofcompositions on the transcription of a mar operon.

The present invention also provides methods for evaluating theantibiotic effectiveness of compositions by assaying their effects uponthe transcription of a mar operon or upon the activity of proteinsencoded by a mar operon. In particular, the present invention providesmethods for assessing the ability or inability of a composition toinhibit the activity of a mar repressor, to enhance the activity of amar repressor, or to inhibit the activity of a mar activator.Compositions which enhance the activity of a mar repressor or inhibitthe activity of a mar activator will be useful either alone or incombination with antibiotics to combat bacteria. A method of treatmentfor bacterial infections using a combination of such compositions alongwith antibiotics is thus provided.

The present invention also provides methods for evaluating theantibiotic effectiveness of compositions by assaying their effects onbacteria in which the expression of a mar operon has been substantiallyincreased and on bacteria in which the expression of a mar operon hasbeen substantially decreased. To this end, methods of producing suchbacteria and such bacteria themselves are provided.

The present invention also provides tests for identifying loci inbacteria which are subject to regulation, directly or indirectly, by amar operon. Because such loci may be involved in the expression of a Marphenotype, their identification will be useful in developing antibioticcompositions which affect the products or expression of those loci.

The present invention also provides cloned bacterial loci and fragmentsthereof which are subject to mar operon regulation and which, therefore,form part of a mar regulon. Using such clones, substantially pureprotein encoded by these loci are provided. In addition, using suchclones, isolated nucleotide sequences, either sense or anti-sense tothese loci, are provided. These sequences are useful as probes forsubstantially homologous loci in other species including bacteria,fungi, parasites, and animal cells and for altering the expression of aMar phenotype in bacteria.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram of the first 3.5 kb of SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms used in biochemistry,molecular biology and recombinant DNA technology are extensivelyutilized. In addition, certain new terms are introduced for greater easeof exposition and to more clearly and distinctly point out the subjectmatter of the invention. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

By a "locus" is understood a nucleotide sequence encoding a peptide. Alocus consists of a start codon, a stop codon and at least one codonencoding an amino acid residue in between. Typically, a locus istranscribed to produce an mRNA transcript and that transcript istranslated to produce a peptide.

By "regulatory region" is understood a nucleotide sequence involved inregulating the transcription of one or more loci. Regulatory regionswill include a promoter sequence at which an RNA polymerase may bindand, typically, an operator sequence which may be bound by a repressorprotein. Additionally, regulatory regions may include enhancers oftranscription.

By "operon" is understood one or more loci operably joined to aregulatory region such that, under appropriate conditions, an RNApolymerase may bind to a promoter sequence in the regulatory region andproceed to transcribe the loci. The loci within an operon share a commonregulatory region and, therefore, are substantially regulated as a unit.Amongst the loci in an operon may be a repressor locus which encodes arepressor protein which, under appropriate conditions, binds to theoperator of the operon so as to substantially decrease expression of theloci in the operon.

By "regulon" is understood two or more loci in two or more differentoperons whose expression is regulated by a common repressor or activatorprotein. A "first" operon may, for example, encode a repressor proteinwhich, under appropriate conditions, binds to the operators of two ormore different operons so as to substantially inhibit transcription ofthe loci within those operons. Or, a "first" operon may encode anactivator protein which interferes with the activity of one or morerepressors of two or more different operons so as to substantiallyincrease the transcription of the loci within those operons.Alternatively, a "first" operon may encode a protein which affects thetranslation or activity of proteins encoded by one or more loci in twoor more different operons. In each of these cases, the latter operonsform a regulon which is regulated by a common protein product of the"first" operon.

By a "bacterial multiple antibiotic resistance regulon" ("mar regulon")is understood a regulon encoding a multiplicity of protein productswhich are regulated in expression or activity by a common proteinproduct and which can cause a substantial increase in resistance to amultiplicity of antibiotics, at least some of which antibiotics areunrelated structurally.

By a "bacterial multiple antibiotic resistance operon" ("mar operon") isunderstood a bacterial operon which, by its expression, affects theexpression of two or more different operons which form a mar regulon.That is, by a "bacterial multiple antibiotic resistance operon" isunderstood a bacterial operon which, by its expression, affects theexpression of two or more loci in two or more different operons, orwhich affects the activity of two or more protein products of such loci,so as to substantially increase resistance to a multiplicity ofantibiotics, at least some of which are structurally unrelated. Amongstthe loci in a bacterial multiple antibiotic resistance operon, there isat least one locus encoding an activator of a bacterial multipleantibiotic resistance regulon. Amongst the loci in a bacterial multipleantibiotic resistance operon, there may also be a locus encoding arepressor of the bacterial multiple antibiotic resistance operon. Themar operon of E. coli includes the marO region and marR, marA and marBloci disclosed herein and is, therefore, also referred to as the marRABoperon.

By a "repressor of a bacterial multiple antibiotic resistance operon"("mar repressor") is understood a protein which, under appropriateconditions, binds to the operator of the operon so as to substantiallyinhibit the transcription of the operon. Such repressor proteins areencoded by repressor loci of bacterial multiple antibiotic resistanceoperons.

By an "activator of a bacterial multiple antibiotic resistance regulon"("mar activator") is understood a protein encoded by a locus within amar operon which, under appropriate conditions, affects the expressionof two or more loci in a mar regulon or the activity of two or moreproteins from such a regulon so as to cause expression of a bacterialmultiple antibiotic resistance phenotype.

By an "enhancer of a bacterial multiple antibiotic resistance regulon"("mar enhancer") is understood a protein encoded by a locus within a maroperon which, under appropriate conditions, enhances the expression oractivity of a mar activator so as to increase expression of a bacterialmultiple antibiotic resistance phenotype.

By a "bacterial multiple antibiotic resistance phenotype" ("Marphenotype") is understood simultaneous and coordinated resistance to amultiplicity of antibiotics, at least some of which are structurallyunrelated, which is substantially increased relative to typical orwild-type bacteria. The antibiotic resistance is simultaneous andcoordinated in that the resistance to the multiplicity of antibioticsincreases or arises simultaneously and may be decreased or lostsimultaneously.

By an "inducer of a bacterial multiple antibiotic resistance operon"("mar inducer") is understood a chemical composition or moiety which,under appropriate conditions, directly or indirectly inhibits thebinding of a repressor of a mar operon to the regulatory region of thatoperon so as to substantially increase the expression of that operonand, consequently, the expression of a multiple antibiotic resistancephenotype.

By a "marker locus" is understood a locus whose expression is easilyassayed. A marker locus is typically a locus encoding an enzyme and theassay may include a substance which changes color in the presence of aproduct of the enzyme's activity. Alternatively, a marker locus mayencode a protein which directly or indirectly affects a visuallyapparent phenotype of an organism such as color or colony type inbacteria. Alternatively, a marker locus may encode a protein whichdirectly or indirectly confers substantial resistance to, sensitivityto, or dependence upon a particular composition.

By "expression" of a locus is understood the transcription of the locusto produce mRNA and the translation of the mRNA transcript to produce apeptide. By "substantially decreased expression of a locus" isunderstood a decrease in detectable expression of its mRNA transcriptand/or protein product of at least about 10% and preferably more than25% of the previous level. By "substantially increased expression of alocus" is understood an increase in the level of its mRNA transcriptand/or protein product of at least about 10% and preferably about 25% ofthe previous level.

By an "operable" locus is understood a locus capable of beingtranscribed under appropriate conditions in vivo or in vitro. A locus ornucleotide sequence is "operably joined" to a regulatory region if,under appropriate conditions, an RNA polymerase may bind to the promoterof the regulatory region and proceed to transcribe the locus ornucleotide sequence in an appropriate reading frame. A locus ornucleotide sequence operably joined to a regulatory region is operable.

A coding sequence and a regulatory region are said to be operably joinedwhen they are covalently linked in such a way as to place expression ofthe coding sequence under the influence or control of the regulatorysequence. Two DNA sequences are said to be operably joined if inductionof the promoter function of one results in the transcription of an mRNAsequence corresponding to the coding sequences of the other. If it isdesired that the RNA transcript be translated into a protein orpolypeptide, there are further considerations. A coding sequencing whichis to be translated into a protein or polypeptide is said to be operablyjoined to a regulatory region if induction of the promoter results inthe transcription of an mRNA transcript corresponding to the codingsequences and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the regulatory sequences to initiate andpromote the transcription of the coding sequences, or (3) interfere withthe ability of the mRNA template to be translated into a functionalprotein. Thus, a regulatory region would be operably joined to a DNAsequence if the promoter were capable of effecting transcription of thatDNA sequence such that the resulting transcript might be translated intoa functional protein or polypeptide.

If it is not desired that the coding sequence be eventually expressed asa protein or polypeptide, as in the case of anti-sense RNA expression,there is no need to ensure that the coding sequences and regulatoryregion are joined without a frame-shift. Thus, a coding sequence whichneed not be eventually expressed as a protein or polypeptide is said tobe operably joined to a regulatory region if induction of promoterfunction results in the transcription of an mRNA sequence correspondingto the coding sequences.

The precise nature of the regulatory region needed for gene expressionmay vary between species or cell types, but shall in general include, asnecessary, 5' non-transcribing and 5' non-translating (non-coding)sequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5' non-transcribing regulatory sequences willinclude a region which contains a promoter for transcriptional controlof the operably joined coding sequences. Such regulatory regions mayalso include enhancer sequences or upstream activator sequences, asdesired.

By "homology" of nucleotide sequences is understood a correlation in thenucleotide composition and ordering of the sequences. If the compositionand ordering of the nucleotides are the same or substantially the same,the sequences are characterized by "sense" homology. If the compositionand ordering of the nucleotides of the sequences are substantiallycomplementary such that the sequences may, under appropriate conditions,hydrogen bond in the manner of complementary strands of DNA, thesequences are characterized by "anti-sense" homology. Sequencescharacterized by sense homology to the mRNA transcript of a locus may,under appropriate conditions, bind to the DNA of that locus so as toinhibit further transcription. Sequences characterized by anti-sensehomology to the mRNA transcript of a locus may, under appropriateconditions, bind to the DNA of that locus so as to inhibit furthertranscription or bind to the mRNA transcript of that locus so as toinhibit translation.

Two nucleotide sequences are substantially homologous if one of them orits anti-sense complement can bind to the other under stricthybridization conditions so as to distinguish that strand from all orsubstantially all other sequences in a cDNA or genomic library.Alternatively, one sequence is substantially homologous to another if itor its anti-sense complement is useful as a probe in screening for thepresence of its homologous DNA or RNA sequence under stricthybridization conditions. "stringent hybridization" conditions is a termof art understood by those of ordinary skill in the art. For any givennucleotide sequence, stringent hybridization conditions are thoseconditions of temperature and buffer solution which will permithybridization of that nucleotide sequence to its complementary sequenceand not to substantially different sequences. The exact conditions whichconstitute "stringent" conditions, depend upon the length of thenucleotide sequence and the frequency of occurrence of subsets of thatsequence within other non-identical sequences. By varying hybridizationconditions from a level of stringency at which no hybridization occursto a level at which hybridization is first observed, one of ordinaryskill in the art can, without undue experimentation, determineconditions which will allow a given sequence to hybridize only withperfectly complementary sequences. Hybridization conditions which permithybridization to imperfectly complementary sequences are employed toisolate nucleotide sequences which are allelic to or evolutionaryhomologs of any given sequence. Suitable ranges of such stringencyconditions are described in Krause, M. H. and S. A. Aaronson, Methods inEnzymology, 200:546-556 (1991). By a sequence which is "substantiallyhomologous" to some specified sequence is understood a sequence whichwill hybridize to the specified sequence, its allelic variants andevolutionary homologs under stringent hybridization conditions so as todistinguish those sequences from non-allelic, non-homologous sequences.

By an "anti-sense locus" is understood a locus which encodes an mRNAtranscript characterized by substantial anti-sense homology to the mRNAencoded by a specified locus. An anti-sense locus to an activator locusof a bacterial multiple antibiotic resistance operon, for example, willencode an mRNA transcript characterized by substantial anti-sensehomology to the mRNA transcript encoded by the activator locus. Theanti-sense mRNA may bind to the DNA of the activator locus so as toinhibit further transcription or it may bind to the mRNA transcript ofthe activator locus so as to inhibit translation.

By "antibiotic" is understood a chemical composition or moiety whichdecreases the viability or which inhibits the growth or reproduction ofmicrobes. As used in this disclosure, for simplicity of exposition,antibiotics are intended to embrace antibacterial, antiviral, antifungaland, generally, antimicrobial compositions.

By an "isolated" nucleotide sequence is understood a nucleotide sequencewhich has been: (1) amplified in vitro by, for example, polymerase chainreaction (PCR); (2) recombinantly produced by cloning; (3) purified, asby cleavage and gel separation; or (4) synthesized by, for example,chemical synthesis. An isolated nucleotide sequence is one which isreadily manipulable by recombinant DNA techniques well known in the art.Thus, a nucleotide sequence contained in a vector in which 5' and 3'restriction cytes are known or for which polymerase chain reaction (PCR)primer sequences have been disclosed is considered isolated, but anucleotide sequence existing in its native state in its natural host isnot. An isolated nucleotide sequence may be substantially purified, butneed not be. For example, a nucleotide sequence that is isolated withina cloning or expression vector is not pure in that it may comprise onlya tiny percentage of the material in the cell in which it resides. Sucha nucleotide sequence is, however, isolated as the term is used hereinbecause it is readily manipulable by standard techniques of recombinantDNA technology known to those of ordinary skill in the art.

By "fragment" is understood a unique fragment, a substantiallycharacteristic fragment, or a functional fragment as defined below.

As used herein, a "unique fragment" of a protein or nucleotide sequenceis a substantially characteristic fragment not currently known to occurelsewhere in nature (except in allelic or allelomorphic variants). Aunique fragment will generally exceed 15 nucleotides or 5 amino acidresidues. One of ordinary skill in the art can substantially identifyunique fragments by searching available computer databases of nucleotideand protein sequences such as Genbank (Los Alamos National Laboratories,USA) or the National Biomedical Research Foundation database. A uniquefragment is particularly useful, for example, in generating monoclonalantibodies or in screening DNA or cDNA libraries.

A "substantially characteristic fragment" of a molecule, such as aprotein or nucleotide sequence, is meant to refer to any portion of themolecule sufficiently rare or sufficiently characteristic of thatmolecule so as to identify it as derived from that molecule or todistinguish it from a class of related molecules. A single amino acid ornucleotide cannot be a substantially characteristic fragment. Asubstantially characteristic fragment of a nucleotide sequence wouldhave utility as a probe in identifying the entire nucleotide sequencefrom which it is derived within a sample of total genomic or DNA. Asubstantially characteristic fragment of a protein would have utility ingenerating antibodies which would distinguish the entire protein fromwhich it is derived from a mixture of many proteins. It is within theknowledge and ability of one ordinarily skilled in the art to recognize,produce and use substantially characteristic fragments as, for example,probes for screening DNA libraries or epitopes for generatingantibodies.

By a "functional fragment" of a molecule is understood a fragmentretaining or possessing substantially the same biological activity asthe intact molecule. For example, a functional fragment of a promotersequence is a nucleotide sequence which retains or possesses the abilityto initiate and promote transcription of a downstream nucleotidesequence by an RNA polymerase. Similarly, a functional fragment of arepressor protein is a fragment which retains or possesses the abilityto bind to an operator sequence of a regulatory region so as tosubstantially inhibit the ability of an RNA polymerase to transcribe thedownstream coding sequences. In all instances, a functional fragment ofa molecule retains at least 10% and at least about 25% of the biologicalactivity of the intact molecule.

The present invention in one aspect provides cloned bacterial multipleantibiotic resistance operons. A bacterial mar operon may be most easilyisolated and cloned from any of a number of species using the nucleotidesequences disclosed herein. Absent the use of the nucleotide sequencesdisclosed herein, a mar operon may still be isolated using the followingprocedures.

A strain of bacteria is first subjected to selection on solid or influid medium containing an antibiotic. The selection may be step-wise,with incrementally increasing concentrations of the antibiotic. Amongstthe surviving cells will be some spontaneous mar operon mutants whichexpress the Mar phenotype. Such Mar mutants may be identified by theircross-resistance to a multiplicity of antibiotics which are structurallyunrelated to the selective agent. The approximate map position of theoperon may then be determined by mating transfer experiments which arewell known in the art.

The Mar phenotype bacteria may then be mutagenized with a transposon andcells reverting to the wild-type (non-Mar) phenotype can be isolated byselecting for antibiotic susceptibility on replicated plates. A highpercentage of the revertants will be the result of inactivation of themar operon by insertion of the transposon within the operon. Using arestriction enzyme known to recognize a site within the transposon,fragments of the transposon joined to segments of chromosomal DNA can becloned into vectors. These clones may, in turn, be used to probe agenomic library of the cells to identify clones bearing at least afragment of the mar operon. Finally, these clones may be tested fortheir ability to complement cells in which there is a large deletionaround the approximate mar map position. Clones capable of providing maroperon activity to the deletants will contain an operable mar operon andmay be sequenced by standard techniques. This technique was used toidentify the mar operon of E. coli (See Example 1). Given the nucleotidesequence of the mar operon of E. coli, however, such a technique is notnecessary for isolating substantially homologous operons in otherspecies.

In one particular aspect, therefore, the present invention provides thecloned wild-type mar operon of E. coli. One sequence containing awild-type mar operon is presented as SEQ ID NO: 1. This sequence hasbeen entered into GenBank with Accession #M96235 and corresponds to the7.8 kbp fragment isolated in Example 1. This sequence or any fragment ofit may, of course, be cloned into any of a number of vectors which areknown to those of ordinary skill in the art.

Conservative variations on the DNA sequence SEQ ID NO: 1 exist whichwill have no substantial effect on the expression of the operon. Thesubstitution of synonymous codons is one example. A small deletion orinsertion which does not disrupt the reading frame is another.Substantially homologous sequences from a mar operon in a species otherthan E. coli are another example, particularly when such operons areidentified by the methods and compositions disclosed herein. Suchconservative variations would be obvious to one of ordinary skill in theart and fall within the spirit and scope of the claims.

The sequence of SEQ ID NO: 1 includes three regions. These regions aredepicted in FIG. 1.

Analysis of this sequence reveals a regulatory region withpromoter-operator sequences, designated maro. The maro sequence extendsfrom approximately nucleotide positions 1234 to 1444 of SEQ ID NO: 1between Region I and Region II. Transcription of Region I proceedsleftward from maro on the DNA strand complementary to SEQ ID NO: 1whereas transcription of Region II proceeds rightward from marO on theDNA strand depicted as SEQ ID NO: 1. The marO sequence includes twopairs of direct repeat (DR) elements. DR-1, 15 bp long with one mismatchat position 9 of the DR (TACTTGCC T/A!GGGCAA), begins at position 1390of SEQ ID NO: 1 and its partner, DR-1', begins at position 1425. DR-1'is part of an imperfect palindrome starting at position 1423(ATTACTTGCCAGGGCAACTAAT) and DR-1 is part of a similar shorterpalindrome. A second DR (DR-2 and DR-2') of 9 bp (GCAACTAAT) flanks onboth sides and partly overlaps the downstream part of DR-1'. For RegionI, marO includes a promoter with -10 and -35 E. coli consensus sequencesat about nucleotide positions 1350 and 1370, respectively, and/or atabout positions 1275 and 1301, respectively. For Region II, marOincludes a promoter with nearly perfect -10 and -35 E. coli consensussequences at about nucleotide positions 1408 and 1384, respectively.

Region II includes four potential open reading frames (ORFs) designatedORF 125, ORF 144, ORF 129 and ORF 72.

ORF 125 begins with an ATG start codon at nucleotide positions 1502-1504of SEQ ID NO: 1 and ends with a TAA stop codon at positions 1877-1879,and encodes a protein of 125 amino acid residues. Based upon sequencingof Mar mutants, ORF 125 was originally considered the mar repressorlocus but, based on fusion-protein studies of binding to marO, describedmore fully below (See Example 10), ORF 125 does not encode the full marrepressor.

ORF 144 begins with a GTG start codon at nucleotide positions 1445-1447of SEQ ID NO: 1, ends with the same TAA stop codon as ORF 125 atpositions 1877-1879 (thereby encompassing all of ORF 125). ORF 144encodes the full mar repressor (144 amino acid residues) and is,therefore, designated the marR locus. The mar repressor regulates notonly transcription of Region II (in which marR is found) but also has aregulatory function for Region I. The protein, MarR, encoded by ORF 144is disclosed as SEQ ID NO: 4. Note that the first GTG codon istranslated as a Met residue.

The second locus of Region II, marA, corresponds to ORF 129 and encodesa protein of 129 amino acids, designated the mar activator. The marAlocus extends from nucleotide 1893 of SEQ ID NO: 1 to nucleotide 2282.The mar activator (MarA) is a 13 kDa polypeptide that shows strongsimilarity to the family of positive regulators that includes regulatorsof carbohydrate metabolism in Escherichia coli (AraC, RhaR, RhaS, andMelR), Erwinia corotovora (AraC), and Pseudomonas putida (XylS);virulence in Yersinia enterocolitica (VirF) and E. coli (Rns); andoxidative stress response in E. coli (SoxS) (Cohen, Hachler and Levy,1993). For example, the MarA protein is strongly similar (42% identical,65% similar) to the SoxS protein which activates the soxRS regulon genes(Amabile-Cuevas and Demple, 1991). The protein, MarA, encoded by ORF 129is disclosed as SEQ ID NO: 5

The third locus of Region II, marB, corresponds to ORF 72 and encodes aprotein of 72 amino acids, designated the mar enhancer. The marB locusextends from nucleotide 2314 of SEQ ID NO: 1 to nucleotide 2531. ThemarB locus is necessary for expression of the full Mar phenotypealthough its precise mode of action remains unclear. The protein, MarB,encoded by ORF 72 is disclosed as SEQ ID NO: 6.

The marO region and the marR, marA and marB loci form the mar operon, ormarRAB operon, of E. coli. The mar repressor acts to repress expressionof the operon by binding at marO. The mar activator acts directly orindirectly to alter the expression of other operons, loci or proteinswhich are part of the mar regulon and which are directly involved withthe expression of the Mar phenotype. The mar enhancer augments theexpression of the Mar phenotype. In addition to these E. coli homologsof the bacterial mar operon, hybridization studies, disclosed more fullybelow, indicate that substantially homologous sequences are included inthe genomes of many other bacterial species and that bacterial maroperons share a common ancestry and evolutionarily conserved structure.In light of the present disclosure, one of ordinary skill in the art mayreadily isolate the mar operons of other species.

Transcription from marO through Region I (leftward with respect to SEQID NO: 1 on the complementary DNA strand) proceeds through two openreading frames designated ORF 64 and ORF 156/157. ORF 64 begins with anATG start codon complementary to the TAC found (reading leftward) atpositions 1233-31 of SEQ ID NO: 1 and ends with a TGA stop codoncomplementary to the ACT at positions 1041-1039. ORF 156/157 beginseither at the GTG complementary to the CAC at positions 1042-1040 or atthe ATG complementary to the TAC at positions 1039-1037, and ends withthe TAA complementary to the ATT at positions 571-569 of SEQ ID NO: 1.The protein encoded by ORF 157 is disclosed as SEQ ID NO: 2. Note thatthe first GTG is translated as Met.

Although the functions of the Region I proteins are not yet known, theyare part of the mar regulon and function directly in the phenotypicexpression of Mar. Mar phenotype mutants produce somewhat higher levelsof Region I mRNA than wild-type cells and clearly more in the presenceof tetracycline. In addition, deletants without both Region I and RegionII show 2-3 fold lower antibiotic resistance than deletants without onlyRegion II (see Example 11).

Region III contains a single significant open reading frame, ORF 266,which encodes a protein of 266 residues. ORF 266 is transcribed in theopposite (leftward) direction from the loci of Region II and from thestrand of DNA complementary to SEQ ID NO: 1. ORF 266 begins with an ATGstart complementary to the TAC at positions 3363-3361 of SEQ ID NO: 1and ends with the TAA stop codon complementary to the ATT at positions2565-2563. The function of the ORF 266 protein is currently unknown andthe level of Region III MRNA transcripts are not detectably affected bythe Mar status of cells or the presence of tetracycline. The proteinencoded by ORF 266 is disclosed as SEQ ID NO: 7.

The present invention thus provides cloned fragments of these variousregions including regulatory regions, protein coding sequences or both.Particular examples include cloned mar regulatory sequences and clonedmar repressor, mar activator, and mar enhancer loci of bacterial maroperons. The particular examples provided are the cloned E. coli marosequences and marR, marA, and marB loci. In addition, the cloned E. coliORF 64 and ORF 157 sequences are provided. Such loci are clonedpreferably so as to be operably joined to a regulatory region so thatthey may be expressed under conditions wherein the regulatory region isnot blocked by a repressor. The loci may be operably joined to a marregulatory region or to other regulatory regions depending upon thedesired manner of regulation and levels of expression. In addition, theinvention provides vectors containing an operably cloned mar repressorlocus without a mar activator locus and with or without a mar enhancerlocus (see Example 2), an operably cloned mar activator locus without amar repressor locus and with or without a mar enhancer locus (seeExample 3), and an operably cloned mar enhancer locus without a marrepressor locus and with or without a mar activator locus (see Example3). Given the sequences disclosed herein, as well as the methodsprovided for identifying substantially homologous mar operons in otherspecies, one of ordinary skill in the art is enabled to produce suchcloned loci. In addition, anti-sense clones of these loci can be aseasily produced and are also an aspect of the present invention.

Conservative variations on SEQ ID NO: 1 exist which will have nosubstantial effect on the expression of these loci. The substitution ofsynonymous codons is one example. A small deletion or insertion whichdoes not disrupt the reading frame is another. A sequence from a maroperon in a species other than E. coli is another example, particularlywhen such a sequence is identified by the methods and compositionsdisclosed herein. Such conservative variations would be obvious to oneof ordinary skill in the art and fall within the spirit and scope of theclaims.

The present invention also provides probes useful in identifying maroperons in species other than E. coli. A cloned mar operon or clonedfragment of a mar operon from one species can be used to screen a DNAlibrary of another species to identify potential mar operons by methodswhich are known to those of ordinary skill in the art. DNA homologous tomarRAB has been found among many members of the Enterobacteriaceaeincluding Klebsiella (Cohen, Yan and Levy, 1993). Similarly, inductionof the Mar phenotype by salicylate and acetyl salicylate has beencommonly observed among 58 clinical enteric isolates tested (Foulds andRosner, personal communication). In Klebsiella, Serratia and Pseudomonascepacia, the salicylate decreased the presence of OmpF-like outermembrane porins (Burns and Clark, 1992; Sawai, Hirano and Yamaguchi,1987). Furthermore, in Klebsiella, salicylates increased resistance tovarious antibiotics (including β-lactams and tetracycline), decreasedresistance to aminoglycosides and decreased the amounts of capsularpolysaccharide (Domenico, Hopkins and Cunha, 1990; Domenico, Landolphiand Cunha, 1991). This indicates that mar operons are involved insalicylate induction of Mar phenotypes in many enterobacteria.

In one preferred embodiment, the cloned E. coli mar operon disclosed asSEQ ID NO: 1 or a fragment thereof is used to produce probes which areradioactively labeled with ³² P (see Example 4). In a particularlypreferred embodiment, the probe is a fragment of the E. coli marA ormarR locus and, most preferably, a unique fragment. Such probes havebeen found to hybridize with DNA extracted from a wide variety ofbacteria and may reveal an ancient and evolutionarily highly conservedfamily of loci and operons in species extending beyond bacteria.

Conservative variations on the disclosed DNA sequence exist which willnot substantially impair the effectiveness of such probes. Thesubstitution of a small percentage of the bases, small insertions, andsmall deletions are examples. Sequences from a mar operon in a speciesother than E. coli are another example, particularly when such operonsare identified by the methods and compositions disclosed herein. Suchconservative variations would be obvious to one of ordinary skill in theart and fall within the spirit and scope of the claims.

The present invention in another aspect provides substantially pure marrepressor protein, substantially pure mar activator protein andsubstantially pure mar enhancer protein. In particular, the inventionprovides substantially pure E. coli mar repressor protein, substantiallypure E. coli mar activator protein and substantially pure E. coli marenhancer protein (see Example 5). Substantially pure proteins aresuitable for protein sequencing and are typically at least 90% pure byweight and preferably at least 95% pure by weight. Given the sequencesdisclosed herein, as well as the methods provided for identifyingsubstantially homologous mar operons in other species, suchsubstantially pure proteins can be produced and isolated by one ofordinary skill in the art (Maniatis, et al. 1982).

The invention further provides a cloned fusion of a mar regulatoryregion and a marker locus. In a preferred embodiment, the mar regulatoryregion is the E. coli marO and the marker locus is the β-galactosidasegene, lacZ (see Example 6). Such a fusion on a vector is useful forassaying the ability or inability of compositions to increase ordecrease the expression of a Mar phenotype, as disclosed below.

The present invention also provides for the creation of bacterialstrains which exhibit the Mar phenotype. In particular, the inventionprovides such strains by genetic manipulation and provides such strainsof E. coli.

In one embodiment, anti-sense to the mar repressor locus is introducedwithin the cells. This may be accomplished by exposing the cells tosingle-stranded nucleotides or nucleotide analogs which enter the cell.The nucleotides are characterized by substantial anti-sense homology toeither the mar repressor locus or its mRNA transcript and are ofsufficient length such that they either inhibit the transcription of themar repressor locus by binding to the mar repressor DNA or they inhibittranslation of the mar repressor by binding to the mRNA transcript ofthe mar repressor locus. More preferably, an operable anti-sense locusis introduced within the cells on a vector. Preferably, the anti-senselocus is operably joined to a strong promoter and on a high copy-numberplasmid such that the anti-sense transcripts are expressed at highlevels. For some uses, as disclosed below, a temperature sensitiveplasmid may be preferred. Given the sequences disclosed herein, as wellas the methods provided for identifying substantially homologous maroperons in other species, one of ordinary skill in the art is enabled toproduce such nucleotide sequences and vectors operably expressing suchsequences.

In a preferred embodiment, a strain is created which has a deletion,insertion or substitution in the chromosomal mar repressor locus suchthat functional mar repressor is not produced but the mar activatorlocus is expressed. In a particularly preferred embodiment, a deletionis introduced. This is achieved by cloning the mar operon into atemperature sensitive plasmid which replicates at lower temperatures butdoes not replicate at higher temperatures. Using appropriate restrictionenzymes, any one of numerous possible deletions is introduced into themar repressor locus on the plasmid. The plasmid is then introduced intobacterial cells and the cells are grown at the lower temperature toallow for homologous recombination to introduce the partially deletedmar repressor locus into the bacterial chromosome. The bacteria are thengrown at the higher temperature so that, at cell division, thetemperature sensitive plasmid is lost from the daughter cells. Cells inwhich the deletion was introduced into the chromosome and in which theactivator is constitutively expressed may then be selected withantibiotics.

In another preferred embodiment, a mar activator locus is introducedwithin the cells on a vector. In one embodiment, the mar activator locusis operably joined to the mar regulatory region on a plasmid which doesnot include an operable mar repressor locus. Homologous recombination isemployed, as disclosed above, to inactivate the mar repressor locus onthe chromosome by partial deletion, insertion, or substitution, so thatfunctional mar repressor is not produced and the plasmid copy of the maractivator locus is expressed. In a most preferred embodiment, the maractivator locus is operably joined to a regulatory region other than themar regulatory region such that it is expressed irrespective of thepresence of the mar repressor and the chromosomal mar repressor locusneed not be inactivated. Preferably, the regulatory region contains astrong promoter. In addition, it is preferred in both embodiments thatthe plasmids be high copy-number plasmids. For some uses, as disclosedherein, it may be preferable that the plasmid be temperature sensitive.Given the sequences disclosed herein, as well as the methods providedfor identifying substantially homologous mar operons in other species,one of ordinary skill in the art is enabled to produce such vectors (seeExamples 2 and 3).

The present invention further provides for the creation of bacterialstrains which exhibit increased sensitivity to antibiotics, relative towild-type cells, because they have at least partially lost the abilityto express a mar activator. In particular, the invention provides suchstrains by genetic manipulation and provides such strains of E. coli.

In one embodiment, anti-sense to the chromosomal mar activator locus maybe introduced within the cells. This may be accomplished by exposing thecells to single-stranded nucleotides or nucleotide analogs which enterthe cell. The nucleotides are characterized by substantial anti-sensehomology to either the mar activator locus or its mRNA transcript andare of sufficient length such that they either inhibit the transcriptionof the chromosomal mar activator locus by binding to the mar activatorDNA or they inhibit translation of the mar activator by binding to themRNA transcript of the mar activator locus. More preferably, an operableanti-sense locus is introduced within the cells on a plasmid.Preferably, the anti-sense locus is operably joined to a strong promoterand on a high copy-number plasmid such that the anti-sense transcriptsare expressed at high levels. For some uses, as disclosed below, atemperature sensitive plasmid may be preferred. Given the sequencesdisclosed herein, as well as the methods provided for identifyingsubstantially homologous mar operons in other species, one of ordinaryskill in the art is enabled to produce such nucleotide sequences andvectors operably expressing such sequences.

In another preferred embodiment, a strain is created which has adeletion, insertion or substitution in the chromosomal mar activatorlocus such that functional mar activator cannot be produced. In anotherembodiment, a chromosomal deletion, insertion or substitution isintroduced which is not in the mar activator locus but which entails aframe-shift upstream of the locus such that functional mar activatorprotein is not produced. In another preferred embodiment, the entire maroperon or a substantial part of it may be deleted (see Example 7). Asdisclosed above, such deletions, insertions or substitutions may beachieved by homologous recombination between the chromosomal mar operonand a properly constructed plasmid. To further increase sensitivity toantibiotics, the E. coli ORF 64 and/or ORF 156/157 loci, or theirhomologs in other species, may be similarly inactivated. Thus, inpreferred embodiments, insertions, deletions, substitutions orframe-shifts are introduced into a bacterial chromosome whichsubstantially decrease expression of ORF 64, ORF 156/157 or theirhomologs.

In a most preferred embodiment, a mar repressor locus is introducedwithin the cells on a vector. In this embodiment, the mar repressorlocus is operably joined to a regulatory region other than a marregulatory region such that it is expressed irrespective of its ownpresence. Preferably, the regulatory region will contain a strongpromoter and the vectors are high copy-number plasmids. For some uses,as disclosed below, it may be preferable that the plasmid be temperaturesensitive. Given the sequences disclosed herein, as well as the methodsprovided for identifying substantially homologous mar operons in otherspecies, one of ordinary skill in the art is enabled to produce suchvectors (see Examples 2 and 3).

The present invention also provides an assay for compositions whichinduce a Mar phenotype by interfering with the activity of a marrepressor or which increase the sensitivity of cells to antibioticcompositions by enhancing the activity of a mar repressor. In eachembodiment, cells which are not characterized by the Mar phenotype areexposed to a composition and then the level of expression of a locusunder the control of a mar regulatory region is assayed. The locus maybe contained within a mar operon or may be operably joined to a marregulatory region and introduced into the cells on a vector.

In one embodiment, the level of a protein product of a locus under thecontrol of a mar regulatory region is directly measured. This may beaccomplished by, for example, polyacrylamide gel electrophoresis or byany of a variety of other means which are well known to those ofordinary skill in the art.

In another embodiment, the levels of the mRNA transcript of a locusunder the control of a mar regulatory region are directly measured. Thismay be accomplished, as is well known in the art, by performing aNorthern hybridization with a probe which has been radioactively labeledwith ³² p and measuring the level of radioactive probe bound. In apreferred embodiment, the locus is a mar operon activator locus (seeExample 8). Probes for such a locus are disclosed above. In addition, inanother preferred embodiment, the locus is at least one of the openreading frames of the E. coli Region I (see FIG. 1) disclosed in SEQ IDNO: 1.

In a preferred embodiment, a fusion of a marker locus to a marregulatory region is introduced within the cells. One such fusion isdisclosed in Example 6. In one embodiment, the marker fusion of Example6 is introduced within cells and the cells are then grown in LB broth at30° C. for one hour in the presence of the compound to be tested. Thelevel of activity of the β-D-galactosidase marker can be determined bythe O-nitrophenyl-β-D-galactoside assay described in Maniatis, et al.(1982).

In each of these embodiments, it is further preferred that a vectorbearing an operable mar repressor locus be introduced within the cells.This will cause increased repression of the mar operon and improve theability of the assay to detect inducers of the Mar phenotype. The marrepressor locus is preferably introduced into the chromosome byhomologous recombination and is operably joined to a regulatory regionother than a mar regulatory region such that it does not repress it ownexpression. Such vectors are disclosed above.

The most preferred embodiment is an assay employing cells into whichhave been introduced both a marker locus fused to a mar regulatoryregion and an operable mar repressor locus which is not controlled by amar regulatory region.

The present invention also provides assays for compositions which act toprevent the expression of a Mar phenotype or which cause cells to becomeeven more sensitive to certain compounds than wild-type cells, by actingas inhibitors of mar operon expression. In each embodiment, cells whichpossess an operable mar activator locus are exposed to a composition andthen the level of expression of a locus, the expression of which is atleast in part controlled by mar operon expression, is assayed. The locusmay be naturally occurring in the cells or may be operably joined to aregulatory region influenced by mar operon expression.

As disclosed above, the assay may be a direct assay for a translationproduct of the locus by, for example, electrophoresis of cellularproteins, for a transcription product of the locus by, for example, aNorthern blot of cellular mRNA or, in a preferred embodiment, the locusis a marker locus, the activity of which is easily assayed.

In a most preferred embodiment, a marker locus is operably joined to theregulatory region of an operon which is affected by mar operonexpression. Means of identifying such loci are disclosed below. Thefusion of the regulatory region and marker is then introduced intocells. After the cells have been exposed to a composition, changes inthe level of expression of the marker locus can be assayed as anindication of the effect of the composition on the expression of the maroperon. In a particularly preferred embodiment, the regulatory region isfrom the micF or ompF loci of E. coli. The expression of the micF locusis affected by mar operon expression. The micF locus, in turn, affectsthe expression of the ompF locus. These loci or their regulatory regionsmay be operably fused to, for example, lacZ and the activity of theβ-D-galactosidase determined as described above. In another preferredembodiment, the regulatory region is marO and the marker locus is one ofthe loci encoded by the E. coli Region I shown in FIG. 1 and disclosedin SEQ ID NO: 1. In this embodiment, the assay is for the transcriptionor translation products of Region I.

The present invention also provides assays for identifying loci involvedin the expression of a Mar phenotype other than mar operons. That is,the invention provides assays for loci whose expression is directly orindirectly regulated by a mar activator protein.

In one embodiment, substantially purified mar activator protein,disclosed above, is mixed with the fragmented genomic DNA of a speciesunder conditions which permit it to bind to appropriate DNA sequences.DNA fragments to which the activator has bound may then be isolated onfilters, in polyacrylamide gels, or by other methods well known to thoseof ordinary skill in the art. Those fragments may then be cloned intovectors and used as probes to locate and isolate their correspondingloci or may be sequenced to identify gene products associated with them.

In another embodiment, a cell line is employed into which has beenintroduced a vector bearing an operable mar activator locus such thatthe cells express the Mar phenotype. Preferably, the activator locus isjoined to a regulatory region other than the mar regulatory region suchthat its level of expression is high. Alternatively, the mar repressorlocus may be inactivated by deletion, insertion or substitution, asdisclosed above and a plasmid bearing an operable activator locus butnot an operable repressor locus, as disclosed above, may be introducedwithin the cells. The total mRNA from these cells may then be comparedto the total mRNA of cells which are not expressing the Mar phenotype.In a most preferred embodiment, this is accomplished by creating a cDNAlibrary of the total mRNA from the mar strain and the non-mar strain.This cDNA library is then used to generate probes to screen, by standardNorthern technique, the total mRNA from the mar strain and the non-marstrain. Any cDNA probes that hybridize to the mRNA of one strain but notto the mRNA of the other will correspond to loci involved in theexpression of the Mar phenotype. Those probes may then be used toidentify such loci by standard techniques. An alternative approach wouldemploy subtractive screening. The cDNA from a strain expressing a maractivator locus can be hybridized to excess mRNA from a strain deletedof that locus. Subsequently, those cDNAs which do not hybridize can beisolated by, for example, hydroxyapatite chromatography and used toidentify mar related loci.

In another embodiment, a promoterless and therefore inoperable markerlocus is introduced into the cell and allowed to insert randomly intothe chromosome. The cells are then manipulated so as to change theirphenotype either from non-mar to mar or from mar to non-mar. Cells inwhich the expression of the marker changes along with the change in Marphenotype, contain markers which have operably inserted into loci whichare regulated directly or indirectly by a mar operon (See Example 9).The two alternative versions of this embodiment are describedseparately, below.

In one version of the above embodiment, the cells do not initiallyexpress the Mar phenotype but contain an operable mar operon which iscapable of being induced. It is particularly preferred that a vectorbearing an operable mar repressor locus, as disclosed above, beintroduced within the cells such that the expression of the maractivator locus is initially minimal. A promoterless marker locuscontained within a transposon and inserted within a phage, for exampleλ::TnphoA or λ::TnlacZ, is introduced into the cells and allowed torandomly integrate into the genome. In addition, it is desirable thatthe transposon also include a locus conferring resistance to kanamycinor another appropriate antibiotic. A number of colonies, preferably atleast two thousand and, more preferably, at least ten thousand, are thenisolated on plates containing kanamycin or another appropriateantibiotic. These colonies are then examined for expression of themarker locus. If the marker is phoA or lacZ and the cells are grown onplates with 5-bromo-4-chloro-3-indolyl phosphate (XP plates) or5-bromo-4-chloro-3-indolyl β-D-galactoside (XG plates), colonies inwhich the marker operably inserted into an actively expressed locus willbe blue whereas colonies in which the marker failed to insert, insertedinoperably, or inserted operably into a repressed locus will appearwhite. The colonies in which the marker is not expressed are thenisolated and the cells are grown in the presence of a known inducer ofthe mar operon (e.g. salicylate or tetracycline for E. coli). Subsequentto such treatment, colonies which express the marker (and, in thisexample, turn blue) are isolated. These colonies contain the markeroperably inserted in a locus that is subject to regulation by a maroperon. The DNA of these colonies may then be fragmented and cloned.Those clones which confer resistance to kanamycin or another appropriateantibiotic will contain the marker in the transposon as well as DNAadjacent to the insertion site. The genomic DNA adjacent to theinsertion site of the transposon can then be isolated and the locus intowhich the transposon inserted can be identified by techniques known tothose of ordinary skill in the art. That locus will, by this method, beidentified as one which is involved in the expression of the Marphenotype.

In a most preferred version of the above embodiment, the cells initiallyexpress the Mar phenotype but can easily be caused to express thenon-Mar phenotype. As above, a promoterless marker in a transposon isintroduced within the cells and allowed to randomly integrate into thechromosome. And, as above, the transposon also encodes a locusconferring resistance to kanamycin or another appropriate antibiotic. Atemperature sensitive plasmid, such as pMAK705 (Hamilton, et al. 1989),bearing an operable mar activator locus is introduced within the cells.The plasmid may bear the activator locus operably joined to a marregulatory region but without the mar repressor locus or, preferably,may contain an activator locus operably joined to a regulatory regionother than a mar regulatory region such that its level of expression ishigh. If the mar activator locus is operably joined to a mar regulatoryregion, the chromosomal mar repressor locus must be inactivated by anyof the means disclosed above. In addition, the chromosomal mar activatorlocus is inactivated by any of the means disclosed above so that theexpression of the Mar phenotype is dependent upon the plasmid copy ofthe activator locus and the cells are recombination deficient (e.g.recA₋₋) so that the activator locus on the plasmid cannot be introducedinto the chromosome. Initially, the cells are grown at a temperature atwhich the temperature sensitive plasmid replicates (e.g. 30° C. forpMAK705) and in the presence of kanamycin or another appropriateantibiotic. In this embodiment, a number of colonies, preferably atleast two thousand and, more preferably, at least ten thousand, are thenisolated and examined for expression of the marker locus. If the markeris phoA or lacZ and the cells are grown on X-P or X-G plates, forexample, colonies in which the marker operably inserted into an activelyexpressed locus will be blue whereas colonies in which the marker failedto insert, inserted inoperably, or inserted operably into a repressedlocus will appear white. The colonies in which the marker is expressedare then isolated and the cells are grown at an elevated temperature(e.g. 42° C. for pMAK705) such that the temperature sensitive plasmidand, consequently, the Mar phenotype are lost. Then, colonies which nolonger express the marker are isolated. These colonies contain themarker in the transposon operably inserted in a locus that is subject toregulation by a mar operon. As described above, the kanamycin or otherresistance locus in the transposon can be used to isolate a fragmentcontaining the transposon and DNA adjacent to the insertion site of thetransposon. The locus into which the transposon inserted can then beidentified by techniques known to those of ordinary skill in the art.That locus will, by this method, be identified as one which is involvedin the expression of the Mar phenotype.

The present invention further provides compositions and a method fortheir use in treating bacterial infections. By employing the assaysdisclosed above, one of ordinary skill in the art is enabled to identifycompositions which inhibit the expression of a bacterial mar operon.These compositions may be administered to a human or other animal alongwith known antibiotics. By inhibiting the expression of the mar operon,these compositions will either enhance the effectiveness of the knownantibiotic or will render an otherwise ineffective antibiotic effective.Such compositions, once identified by the means disclosed herein, can becombined in pharmaceutically effective amounts with known antibiotics byone ordinarily skilled in the art.

EXAMPLE 1

A mar mutant of E. coli K12 designated AG102 was derived from awild-type strain designated AG100 by selection with antibiotics (seeGeorge and Levy, 1983b). AG102 was then subjected to λ b221 c1857rex::Tn5 mutagenesis. A revertant from the Mar phenotype, resulting fromTn5 insertion within the mar operon, was isolated and designated AG1025.Exploiting the single BamHI site in Tn5, the AG1025 chromosomal DNA wasdigested with BamHI or partially digested with Sau3A. The resultingfragments were ligated into the single BamHI site of the highcopy-number plasmid vector pUC18. Two clones, designated pKanl and pKan2(see Hachler, Cohen and Levy, 1991), were isolated which contained the3.2 kbp of Tn5 upstream of its internal BamHI site. A 2 kbp HpaIfragment of pKanl, containing only 187 bp from the IS50L of Tn5 and 1.85kbp of chromosomal DNA was used as a probe to screen a λ phasmid libraryderived from partial Sau3A digests of the E. coli K12 derivative W3110(see Elledge and Walker, 1985). Isolation was performed in host strainPLK1738 in which the marA region has been deleted. Two phasmidsidentified by the probe were introduced by transduction into a deletionstrain HH84 in which the region including the mar operon had beendeleted from the chromosome. These phasmids were capable of restoringmar operon activity. One of the fragments, 13.1 kbp in length, was usedfor subcloning into the low copy-number vector pHSG415. mar activity wastested in CH164, a ΔmarA strain genetically related to the originalAG100 mar mutants. Subclones containing either the 9 kbp PstI or 7.8 kbpHpaI-PstI fragment, but none containing any smaller fragments, producedMar mutants. (For detail on the genotypes of the strains and vectors,see Hachler, Cohen, and Levy, 1991, incorporated herein by reference.)

EXAMPLE 2

The mar repressor locus, marR, was cloned from the wild-type plasmidpHHM183 (see Hachler, Cohen and Levy, 1991) as an 818 bp DraI fragmentinto the SmaI site of the high copy-number cloning vector pUC18. Theplasmid was designated p125WT. A mutant mar repressor locus causingexpression of the Mar phenotype was cloned on a 850 bp DraI-HpaIfragment from plasmid pKanl (see Hachler, Cohen and Levy, 1991) intopUC18. This plasmid was designated p125mar. These plasmids have beenintroduced into a number of E. coli K12 strains such as the wild-typeAG100 and mar mutant AG102. The full genotypes of these strains may befound in Cohen et al., 1988, incorporated herein by reference.

EXAMPLE 3

The entire E. coli mar operon, marA, and marB have been cloned intovarious vectors and introduced within various hosts using the disclosedsequences and the polymerase chain reaction to generate the fragmentsdisclosed below. In particular, for cloning the entire operon, the PCRprimers at the 5' end were nucleotides 1311-1328 of SEQ ID NO: 1 andnucleotides 2575-2592 at the 3' end. In addition PstI linkers wereincluded at both ends. For cloning the marA locus, the PCR primers werenucleotides 1893-1908 at the 5' end and nucleotides 2265-2282 at the 3'end with EcoRI linkers at both ends. For cloning the marB locus, the PCRprimers were nucleotides 2314-2331 at the 5' end and nucleotides2515-2532 at the 3' end with EcoRI linkers at both ends. The PCRsynthesized genes have been cloned into several plasmid vectors atappropriate single restriction enzyme sites: pUC18, a multicopy ColElderivative; pMAK705, a temperature-sensitive plasmid described inHamilton et al., 1989; and pMAL-C2, a plasmid used for expressing theprotein fused to MalE (New England BioLabs, Beverly, Mass., product#800). The plasmids have been introduced in wild-type E. coli K12;AG102, a mar mutant described in George and Levy, 1983b; AG1025, amarA::Tn5 mar revertant described in George and Levy 1983b; and CH164, amar deleted strain described in Hachler, Cohen and Levy, 1991.

EXAMPLE 4

Cloned copies of SEQ ID NO: 1 were digested with BspHI. A resulting 1.24kbp fragment, corresponding to nucleotides 1073 to 2314 of SEQ ID NO: 1was used to produce ³² P labelled probes. DNA extracted from a largenumber of bacterial species were tested for homology to this probe understringent DNA::DNA hybridization techniques using dot blots and Southernhybridization methods, as are well known to those of ordinary skill inthe art (see, e.g., Maniatis, Fritch and Sambrook, 1982, incorporatedherein by reference). DNA from the following gram-negative genera werefound to hybridize with the probe: Citrobacter, Enterobacter,Escherichia, Hafnia, Klebsiella, Salmonella, and Shigella. Two species,Enterobacter agglomerans and Salmonella sp., were further tested. Thesewere found to produce Mar phenotype mutants when selected by the sameregime employed with E. coli and to produce a 1.4 kb mRNA transcript atheightened levels. The 1.4 kb transcripts were the same size as andhomologous to the mRNA produced at heightened levels in E. coliexpressing the Mar phenotype.

EXAMPLE 5

The E. coli marA fragment described in Example 3 was cloned intopMAL-C2, a vector bearing the maltose binding protein locus, MalE. Thisvector is commercially available as part of a kit for proteinpurification ("Protein fusion and purification system," New EnglandBioLabs, Beverly, Mass., product #800). The clone, including the marAfragment, encoded a fusion product comprising the mar activator proteinand the maltose binding protein linked by a peptide which is cleavableby protease Xa. The fusion protein was made in E. coli TB1 (ara, Δ(lacpro AB) rpsL (Φ80 lacZ ΔM15) hsdR). The fusion protein was thensubstantially purified by amylose column chromatography. The peptidelinking the mar activator protein and the maltose binding protein wascleaved and the substantially purified mar protein collected.

EXAMPLE 6

A 405 bp ThaI fragment containing the E. coli marO region was ligatedinto the SmaI site of pMLB1109, a lacZ transcriptional fusion plasmid.The resulting plasmid construct had lacZ gene expression under thecontrol of the marO promoter. The fusion was introduced into thechromosome of a wild type cell and a mar operon deleted strain. This wasaccomplished by first introducing the marO-lacZ region of the fusionplasmids, by homologous recombination, into the genome of phage λRZ5 byinfecting an E. coli K12 strain designated SPC103 (M4100 (Δlac U169araD, rpsL, relA, thi, fibB) deleted of 39 kbp surrounding the maroperon) bearing one of the fusion plasmids, with λRZ5. The resultinglysate was used to transduce plasmid-less SPC103. Amp^(R), Lac⁺ lysogenswere selected on LB agar containing ampicillin (50 μg/ml) and5-bromo-4-chloro-3-indolyl β-D-galactosidase. Lysates from thesepurified lysogens were then used to infect E. coli MC4100 or SPC103 andAmp^(R), Lac₊ lysogens were again isolated. The resulting strains,SPC104, SPC105, SPC106, and SPC107 were confirmed to have a single copyof the fusion region located in the same site on the chromosome (likelythe att site) by Southern hybridization of PstI-digested chromosomal DNAfrom the strains with a 405 bp EcoRI/BamHI fragment. Lysogens of MC4100(SPC104, SPC105) had 2 bands which hybridized with a 9 kb fragmentrepresenting the naturally occurring marO-marA sequence and a largerfragment (>15 kb) representing the insertion of the maro-lacZ fusionphage into the chromosome. The lysogens of the mar deletion strainSPC103 (SPC106, SPC107) contained only chromosomal sequencescorresponding to the larger band. DNA manipulations and analyses wereperformed according to Maniatis et al. (1982). Assays forβ-galactosidase activity were performed on cells grown for one hour tomid-logarithmic phase in LB broth in the presence of known and potentialinducers of the Mar phenotype at 30° C. The β-galactosidase activity ofthese cells was compared to cells grown similarly but in the absence ofknown inducers of the Mar phenotype.

EXAMPLE 7

A 9 kbp PstI fragment of E. coli K12 chromosomal DNA containing the maroperon was cloned into temperature-sensitive plasmid pMAK705. Theplasmid replicates at 30° C. but is lost from daughter cells during celldivision at 42° C. Using BspHI, a 1.24 kbp deletion corresponding tonucleotide 1073 to nucleotide 2314 of SEQ ID NO: 1 and including all ofthe marO region, the marR and marA loci, and part of the marB locus wasmade within the mar operon on the plasmid. The plasmid was thenintroduced within E. coli K12 AG100 (see George and Levy, 1983b forgenotype information). The plasmid and chromosome were then allowed toundergo homologous recombination and the cells were cured of the plasmidat 42° C. A recombinant strain with the 1.24 kbp deletion in thechromosomal mar operon was isolated and designated AG100 Δ15.

EXAMPLE 8

About 10⁸ E. coli which were not expressing the Mar phenotype were grownat 30° C., collected at the end of logarithmic phase and resuspended infresh broth containing salicylate, a known mar operon inducer, for onehour at 30° C. The mRNA was extracted from these cells and separated bygel electrophoresis. A 1.24 kb BspHI fragment from within the mar operonwhich includes the marA locus was labeled with ³² p by random primingand used as a probe in a Northern hybridization to assay for increasedexpression of the marA locus.

EXAMPLE 9

TnphoA and TnlacZ were used to mutagenize a recA E. coli strain whichhad been deleted of the mar operon and transformed with atemperature-sensitive (curable) plasmid containing the constitutivelyexpressed mar operon. From a total of 2100 fusions, 5 mar-regulatedmutants were identified. Two lacZ fusions showed loss of LacZ activityupon loss of the plasmid at 42° C., while three phoA fusions showed anincrease in PhoA activity with plasmid loss. The DNA sequence of thechromosomal DNA proximal to each of the fusions did not show homologywith any known genes of E. coli. The lacZ fusions were at 31.5 and 14min; two of the phoA fusions were at 77 min and one was at 51.6 min. Inone of the two phoA fusions at 77 min, PhoA activity was associated withthe membranes. This approach has identified new genes in E. coli whichare regulated by the marRAB operon and involved in the Mar phenotype.

EXAMPLE 10

Based on sequencing of Mar mutants, the protein products of both ORF 125and ORF 144 were considered candidates for the mar repressor. Toinvestigate this, fusion proteins of the maltose binding protein (MBP)and each of the two potential repressors, MarR125 and MarR144, wereproduced. MBP-MarR144, but not MBP-MarR125, repressed expression of LacZfrom a marO-lacZ fusion. The fusion proteins MBP-MarR125 and MBP-MarR144were purified by amylose affinity chromatography. Gel retardationstudies showed that purified MBP-MarR144 bound to marO with an affinityof 5×10⁹ M. No binding was seen with MBP-MarR125. Therefore theN-terminal amino acid residues lacking in MBP-MarR125 are required formarO binding. Structurally unrelated compounds (tetracycline,chloramphenicol, ampicillin, DNP and salicylate) at differentconcentrations caused reversal of the binding of MarR (i.e.,MBP-MarR144) to marO.

EXAMPLE 11

In conjunction with SEQ ID NO: 1, the polymerase chain reaction (PCR)was employed to amplify the coding sequences of Region I and Region II.PCR primers were created to flank the coding regions of ORF 156/157, ORF64, Region I, Region II, and Regions I and II together. For cloning ORF156/157, the PCR primers were nucleotides 570-587 at the 5' end of SEQID NO: 1 and nucleotides 1022-1039 at the 3' end with either PstI orEcoRI linkers at the ends. For cloning ORF 64, the PCR primers werenucleotides 1039-1056 at the 5' end and nucleotides 1216-1233 at the 3'end with either PstI or EcoRI linkers at the ends. For cloning theentire Region I sequence, the PCR primers were nucleotides 163-180 atthe 5' end and nucleotides 1216-1233 at the 3' end with either PstI orEcoRI linkers at the ends. For cloning Regions I and II together, thePCR primers were nucleotides 163-180 at the 5' end and nucleotides2575-2592 at the 3' end with PstI linkers at both ends. The PCRsynthesized sequences were cloned into several plasmid vectors atappropriate restriction enzyme sites: pMAK705, a temperature-sensitivelow copy-number plasmid (Hamilton et al. 1989) and pMAL-C2, a plasmidused for expressing the protein fused to MalE (New England BioLabs,Beverly, Mass.).

Plasmid constructs containing different PCR fragments of Region I andRegion II were used in complementation analyses to define the genesrequired to restore multidrug resistance (to tetracycline,chloramphenicol, nalidixic acid, norfloxacin, and rifampicin) in mardeletion and inactivated strains. In two deletion mutants (Δ39 kbincluding mar-MCH164; Δ1.2 kb in the mar operon-WY100) plasmidscontaining marA alone restored wild-type MICs to tetracycline, nalidixicacid, and chloramphenicol. The addition of marB to marA increased theresistance to the drugs 19-46% (depending upon the drug tested),suggesting that both marA and marB are associated with intrinsic drugsusceptibility/resistance.

Plasmids containing both Region I and Region II in the same mutantstrains further increased antibiotic resistance levels 2-3 fold tolevels comparable with Mar mutants. These findings indicate that bothRegion I and Region II are involved with the multiple antibioticresistance phenotype. More detailed results are shown below forcomplementation tests with nalidixic acid (nal), tetracycline (tet) andchloramphenicol (cml).

    ______________________________________                                                         MIC (μg/ml)                                               E. coli strain     nal      Tet    clm                                        ______________________________________                                        AG100 (wild-type)  4.2      3.3    4.6                                        MCH164 (Δ39kb)                                                                             2.2      1.9    0.9                                        MCH164pMAL-marA    5.3      3.4    4.9                                        MCH164pMAL-marB    2.1      2.1    1.0                                        MCH164pMAL-marAB   6.3      4.7    7.2                                        MCH164pMAK-Region II                                                                             6.8      4.7    N/A*                                       MCH164pMAK-Regions I & II                                                                        11.3     10.4   N/A*                                       MCH164pHH193-SEQ ID NO: 1                                                                        13.6     13.1   N/A*                                       AG102 (Mar mutant) 14.5     13.8   >25                                        ______________________________________                                         *Plasmid confers resistance to cml.                                      

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7876 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Escherichia coli                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (572..1042)                                          (D) OTHER INFORMATION: /gene= "ORF156/157"                                    /note= "Start codon indefinite. ORF 157 at                                    complement 572..1042. ORF 156 at complement                                   572..1039. For ORF 157, first GTG encodes Met."                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (1042..1233)                                         (D) OTHER INFORMATION: /gene= "ORF 64"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1445..1876                                                      (D) OTHER INFORMATION: /gene= "marR"                                          /transl.sub.-- except= (pos: 1445 .. 1447, aa: Met)                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1893..2279                                                      (D) OTHER INFORMATION: /gene= "marA"                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2314..2529                                                      (D) OTHER INFORMATION: /gene= "marB"                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: complement (2566..3363)                                         (D) OTHER INFORMATION: /gene= "ORF 266"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: promoter                                                        (B) LOCATION: 1234..1444                                                      (D) OTHER INFORMATION: /standard.sub.-- name= "marO"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTTAACTGTGGTGGTTGTCACCGCCCATTACACGGCATACAGCTATATCGAGCCTTTTGT60                ACAAAACATTGCGGGATTCAGCGCCAACTTTGCCACGGCATTACTGTTATTACTCGGTGG120               TGCGGGCATTATTGGCAGCGTGATTTTCGGTAAACTGGGTAATCAGTATGCGTCTGCGTT180               GGTGAGTACGGCGATTGCGCTGTTGCTGGTGTGCCTGGCATTGCTGTTACCTGCGGCGAA240               CAGTGAAATACACCTCGGGGTGCTGAGTATTTTCTGGGGGATCGCGATGATGATCATCGG300               GCTTGGTATGCAGGTTAAAGTGCTGGCGCTGGCACCAGATGCTACCGACGTCGCGATGGC360               GCTATTCTCCGGCATATTTAATATTGGAATCGGGGCGGGTGCGTTGGTAGGTAATCAGGT420               GAGTTTGCACTGGTCAATGTCGATGATTGGTTATGTGGGCGCGGTGCCTGCTTTTGCCGC480               GTTAATTTGGTCAATCATTATATTTCGCCGCTGGCCAGTGACACTCGAAGAACAGACGCA540               ATAGTTGAAAGGCCCATTCGGGCCTTTTTTAATGGTACGTTTTAATGATTTCCAGGATGC600               CGTTAATAATAAACTGCACACCCATACATACCAGCAGGAATCCCATCAGACGGGAGATCG660               CTTCAATGCCACCCTTGCCCACCAGCCGCATAATTGCGCCGGAGCTGCGTAGGCTTCCCC720               ACAAAATAACCGCCACCAGGAAAAAGATCAGCGGCGGCGCAACCATCAGTACCCAATCAG780               CGAAGGTTGAACTCTGACGCACTGTGGACGCCGAGCTAATAATCATCGCTATGGTTCCCG840               GACCGGCAGTACTTGGCATTGCCAGCGGCACAAAGGCAATATTGGCACTGGGTTCATCTT900               CCAGCTCTTCCGACTTGCTTTTCGCCTCCGGTGAATCAATCGCTTTCTGTTGCGGAAAGA960               GCATCCGAAAACCGATAAACGCGACGATTAAGCCGCCTGCAATTCGCAGACCGGGAATCG1020              AAATGCCAAATGTATCCATCACCAGTTGCCCGCGTAATACGCCACCATCATGATGGCAAA1080              TACGTACACCGAGGCCATCAACGACTGACGATTACGTTCGGCACTGTTCATGTTGCCTGC1140              CAGGCCAAGAAATAACGCGACAGTTGTTAATGGGTTAGCTAACGGCAGCAACACCACCAG1200              CCCCAGGCCAATTGCTTTAAACAAATCTAACATTGGTGGTTGTTATCCTGTGTATCTGGG1260              TTATCAGCGAAAAGTATAAGGGGTAAACAAGGATAAAGTGTCACTCTTTAGCTAGCCTTG1320              CATCGCATTGAACAAAACTTGAACCGATTTAGCAAAACGTGGCATCGGTCAATTCATTCA1380              TTTGACTTATACTTGCCTGGGCAATATTATCCCCTGCAACTAATTACTTGCCAGGGCAAC1440              TAATGTGAAAAGTACCAGCGATCTGTTCAATGAAATTATTCCATTGGGT1489                         MetLysSerThrSerAspLeuPheAsnGluIleIleProLeuGly                                 151015                                                                        CGCTTAATCCATATGGTTAATCAGAAGAAAGATCGCCTGCTTAACGAG1537                          ArgLeuIleHisMetValAsnGlnLysLysAspArgLeuLeuAsnGlu                              202530                                                                        TATCTGTCTCCGCTGGATATTACCGCGGCACAGTTTAAGGTGCTCTGC1585                          TyrLeuSerProLeuAspIleThrAlaAlaGlnPheLysValLeuCys                              354045                                                                        TCTATCCGCTGCGCGGCGTGTATTACTCCGGTTGAACTGAAAAAGGTA1633                          SerIleArgCysAlaAlaCysIleThrProValGluLeuLysLysVal                              505560                                                                        TTGTCGGTCGACCTGGGAGCACTGACCCGTATGCTGGATCGCCTGGTC1681                          LeuSerValAspLeuGlyAlaLeuThrArgMetLeuAspArgLeuVal                              657075                                                                        TGTAAAGGCTGGGTGGAAAGGTTGCCGAACCCGAATGACAAGCGCGGC1729                          CysLysGlyTrpValGluArgLeuProAsnProAsnAspLysArgGly                              80859095                                                                      GTACTGGTAAAACTTACCACCGGCGGCGCGGCAATATGTGAACAATGC1777                          ValLeuValLysLeuThrThrGlyGlyAlaAlaIleCysGluGlnCys                              100105110                                                                     CATCAATTAGTTGGCCAGGACCTGCACCAAGAATTAACAAAAAACCTG1825                          HisGlnLeuValGlyGlnAspLeuHisGlnGluLeuThrLysAsnLeu                              115120125                                                                     ACGGCGGACGAAGTGGCAACACTTGAGTATTTGCTTAAGAAAGTCCTG1873                          ThrAlaAspGluValAlaThrLeuGluTyrLeuLeuLysLysValLeu                              130135140                                                                     CCGTAAACAAAAAAGAGGTATGACGATGTCCAGACGCAATACTGACGCT1922                         ProMetThrMetSerArgArgAsnThrAspAla                                             1510                                                                          ATTACCATTCATAGCATTTTGGACTGGATCGAGGACAACCTGGAATCG1970                          IleThrIleHisSerIleLeuAspTrpIleGluAspAsnLeuGluSer                              152025                                                                        CCACTGTCACTGGAGAAAGTGTCAGAGCGTTCGGGTTACTCCAAATGG2018                          ProLeuSerLeuGluLysValSerGluArgSerGlyTyrSerLysTrp                              303540                                                                        CACCTGCAACGGATGTTTAAAAAAGAAACCGGTCATTCATTAGGCCAA2066                          HisLeuGlnArgMetPheLysLysGluThrGlyHisSerLeuGlyGln                              455055                                                                        TACATCCGCAGCCGTAAGATGACGGAAATCGCGCAAAAGCTGAAGGAA2114                          TyrIleArgSerArgLysMetThrGluIleAlaGlnLysLeuLysGlu                              606570                                                                        AGTAACGAGCCGATACTCTATCTGGCAGAACGATATGGCTTCGAGTCG2162                          SerAsnGluProIleLeuTyrLeuAlaGluArgTyrGlyPheGluSer                              75808590                                                                      CAACAAACTCTGACCCGAACCTTCAAAAATTACTTTGATGTTCCGCCG2210                          GlnGlnThrLeuThrArgThrPheLysAsnTyrPheAspValProPro                              95100105                                                                      CATAAATACCGGATGACCAATATGCAGGGCGAATCGCGCTTTTTACAT2258                          HisLysTyrArgMetThrAsnMetGlnGlyGluSerArgPheLeuHis                              110115120                                                                     CCATTAAATCATTACAACAGCTAGTTGAAAACGTGACAACGTCACTGAGGC2309                       ProLeuAsnHisTyrAsnSer                                                         125                                                                           AATCATGAAACCACTTTCATCCGCAATAGCAGCTGCGCTTATTCTCTTT2358                         MetLysProLeuSerSerAlaIleAlaAlaAlaLeuIleLeuPhe                                 151015                                                                        TCCGCGCAGGGCGTTGCGGAACAAACCACGCAGCCAGTTGTTACTTCT2406                          SerAlaGlnGlyValAlaGluGlnThrThrGlnProValValThrSer                              202530                                                                        TGTGCCAATGTCGTGGTTGTTCCCCCATCGCAGGAACACCCACCGTTT2454                          CysAlaAsnValValValValProProSerGlnGluHisProProPhe                              354045                                                                        GATTTAAATCACATGGGTACTGGCAGTGATAAGTCGGATGCGCTCGGC2502                          AspLeuAsnHisMetGlyThrGlySerAspLysSerAspAlaLeuGly                              505560                                                                        GTGCCCTATTATAATCAACACGCTATGTAGTTTGTTCTGGCCCCGAC2549                           ValProTyrTyrAsnGlnHisAlaMet                                                   6570                                                                          ATCTCGGGGCTTATTAACTTCCCACCTTTACCGCTTTACGCCACCGCAAGCCAAATACAT2609              TGATATACAGCCCGGTCATAATGAGCACCGCACCTAAAAATTGCAGACCCGTTAAGCGTT2669              CATCCAACAATAGTGCCGCACTTGCCAGTCCTACTACGGGCACCAGTAACGATAACGGTG2729              CAACCCGCCAGGTTTCATAGCGTCCCAGTAACGTCCCCCAGATCCCATAACCAACAATTG2789              TCGCCACAAACGCCAGATACATCAGAGACAAGATGGTGGTCATATCGATAGTAACCAGAC2849              TGTGAATCATGGTTGCGGAACCATCGAGAATCAGCGAGGCAACAAAGAAGGGAATGATTG2909              GGATTAAAGCGCTCCAGATTACCAGCGACATCACCGCCGGACGCGTTGAGTGCGACATGA2969              TCTTTTTATTGAAGATGTTGCCACACGCCCAACTAAATGCTGCCGCCAGGGTCAACATAA3029              AGCCGAGCATCGCCACATGCTGACCGTTCAGACTATCTTCGATTAACACCAGTACGCCAA3089              AAATCGCTAAGGCGATCCCCGCCAATTGTTTGCCATGCAGTCGCTCCCCGAAAGTAAACG3149              CGCCAAGCATGATAGTAAAAAACGCCTGTGCCTGTAACACCAGCGAAGCCAGTCCAGCAG3209              GCATACCGAAGTTAATGGCACAAAAAAGAAAAGCAAACTGCGCAAAACTGATGGTTAATC3269              CATACCCCAGCAGCAAATTCAGTGGTACTTTCGGTCGTGCGACAAAAAAGATAGCCGGAA3329              AAGCGACCAGCATAAAGCGCAAACCGGCCAGCATCAGCGTGGCATGTTATGAAGCCCCAC3389              TTTGATGACCACAAAATTTAGCCCCCATACGACCACTACCAGTAGCGCCAACACCCCATC3449              TTTTCGCGACATTCTACCGCCTCTGAATTTCATCTTTTGTAAGCAATCAACTTAGCTGAA3509              TTTACTTTTCTTTAACAGTTGATTCGTTAGTCGCCGGTTACGACGGCATTAATGCGCAAA3569              TAAGTCGCTATACTTCGGATTTTTGCCATGCTATTTCTTTACATCTCTAAAACAAAACAT3629              AACGAAACGCACTGCCGGACAGACAAATGAACTTATCCCTACGACGCTCTACCAGCGCCC3689              TTCTTGCCTCGTCGTTGTTATTAACCATCGGACGCGGCGCTACCGTGCCATTTATGACCA3749              TTTACTTGAGTCGCCAGTACAGCCTGAGTGTCGATCTAATCGGTTATGCGATGACAATTG3809              CGCTCACTATTGGCGTCGTTTTTAGCCTCGGTTTTGGTATCCTGGCGGATAAGTTCGACA3869              AGAAACGCTATATGTTACTGGCAATTACCGCCTTCGCCAGCGGTTTTATTGCCATTACTT3929              TAGTGAATAACGTGACGCTGGTTGTGCTCTTTTTTGCCCTCATTAACTGCGCCTATTCTG3989              TTTTTGCTACCGTGCTGAAAGCCTGGTTTGCCGACAATCTTTCGTCCACCAGCAAAACGA4049              AAATCTTCTCAATCAACTACACCATGCTAAACATTGGCTGACCATCGGTCCGCCGCTCGG4109              CACGCTGTTGGTAATGCAGAGCATCAATCTGCCCTTCTGGCTGGCAGCTATCTGTTCCGC4169              GTTTCCCATGCTTTTCATTCAAATTTGGGTAAAGCGCAGCGAGAAAATCATCGCCACGGA4229              AACAGGCAGTGTCTGGTCGCCGAAAGTTTTATTACAAGATAAAGCACTGTTGTGGTTTAC4289              CTGCTCTGGTTTTCTGGCTTCTTTTGTAAGCGGCGCATTTGCTTCATGCATTTCACAATA4349              TGTGATGGTGATTGCTGATGGGGATTTTGCCGAAAAGGTGGTCGCGGTTGTTCTTCCGGT4409              GAATGCTGCCATGGTGGTTACGTTGCAATATTCCGTGGGCCGCCGACTTAACCCGGCTAA4469              CATCCGCGCGCTGATGACAGCAGGCACCCTCTGTTTCGTCATCGGTCTGGTCGGTTTTAT4529              TTTTTCCGGCAACAGCCTGCTATTGTGGGGTATGTCAGCTGCGGTATTTACTGTCGGTGA4589              AATCATTTATGCGCCGGGCGAGTATATGTTGATTGACCATATTGCGCCGCCAGAAATGAA4649              AGCCAGCTATTTTTCCGCCCAGTCTTTAGGCTGGCTTGGTGCCGCGATTAACCCATTAGT4709              GAGTGGCGTAGTGCTAACCAGCCTGCCGCCTTCCTCGCTGTTTGTCATCTTAGCGTTGGT4769              GATCATTGCTGCGTGGGTGCTGATGTTAAAAGGGATTCGAGCAAGACCGTGGGGGCAGCC4829              CGCGCTTTGTTGATTTAAGTCGAACACAATAAAGATTTAATTCAGCCTTCGTTTAGGTTA4889              CCTCTGCTAATATCTTTCTCATTGAGATGAAAATTAAGGTAAGCGAGGAAACACACCACA4949              CCATAAACGGAGGCAAATAATGCTGGGTAATATGAATGTTTTTATGGCCGTACTGGGAAT5009              AATTTTATTTTCTGGTTTTCTGGCCGCGTATTTCAGCCACAAATGGGATGACTAATGAAC5069              GGAGATAATCCCTCACCTAACCGGCCCCTTGTTACAGTTGTGTACAAGGGGCCTGATTTT5129              TATGACGGCGAAAAAAAACCGCCAGTAAACCGGCGGTGAATGCTTGCATGGATAGATTTG5189              TGTTTTGCTTTTACGCTAACAGGCATTTTCCTGCACTGATAACGAATCGTTGACACAGTA5249              GCATCAGTTTTCTCAATGAATGTTAAACGGAGCTTAAACTCGGTTAATCACATTTTGTTC5309              GTCAATAAACATGCAGCGATTTCTTCCGGTTTGCTTACCCTCATACATTGCCCGGTCCGC5369              TCTTCCAATGACCACATCCAGAGGCTCTTCAGGAAATGCGCGACTCACACCTGCTGTCAC5429              GGTAATGTTGATATGCCCTTCAGAATGTGTGATGGCATGGTTATCGACTAACTGGCAAAT5489              TCTGACACCTGCACGACATGCTTCTTCATCATTAGCCGCTTTGACAATAATGATAAATTC5549              TTCGCCCCCGTAGCGATAAACCGTTTCGTAATCACGCGTCCAACTGGCTAAGTAAGTTGC5609              CAGGGTGCGTAATACTACATCGCCGATTAAATGCCCGTAGTATCATTAACCAATTTAAAT5669              CGGTCAATATCCAACAACATTAAATAAAGATTCAGAGGCTCAGCGTTGCGTAACTGATGA5729              TCAAAGGATTCATCAAGAACCCGACGACCCGGCAATCCCGTCAAAACATCCATATTGCTA5789              CGGATCGTCAGCAAATAAATTTTGTAATCGGTTAATGCCGCAGTAAAAGAAAGCAACCCC5849              TCCTGAAAGGCGTCGAAATGCGCGTCCTGCCAGTGATTTTCAACAATAGCCAGCATTAAT5909              TCCCGACCACAGTTATGCATATGTTGATGGGCAGAATCCATTAGCCGAACGTAAGGTAAT5969              TCATCGTTATCGAGTGGCCCCAGATGATCAATCCACCGACCAAACTGGCACAGTCCATAA6029              GAATGGTTATCCGTTATTTCTGGCTTACTGGCATCTCTCGCGACCACGCTGTGAAACATA6089              CTCACCAGCCACTGGTAGTGGGCATCGATAGCCTTATTGAGATTTAACAAGATGGCATCA6149              ATTTCCGTTGTCTTCTTGATCATTGCCACTCCTTTTTCACAGTTCCTTGTGCGCGCTATT6209              CTAACGAGAGAAAAGCAAAATTACGTCAATATTTTCATAGAAATCCGAAGTTATGAGTCA6269              TCTCTGAGATAACATTGTGATTTAAAACAAAATCAGCGAATAAAAAAGTGTTTAATTCTG6329              TAAATTACCTCTGCATTATCGTAAATAAAAGGATGACAAATAGCATAACCCAATACCCTA6389              ATGGCCCAGTAGTTCAGGCCATCAGGCTAATTTATTTTTATTTCTGCAAATGAGTGACCC6449              GAACGACGGCCGGCGCGCTTTTCTTATCCAGACTGCCACTAATGTTGATCATCTGGTCCG6509              GCTGAACTTCTCGTCCATCAAAGACGGCCGCAGGAATAACGACATTAATTTCACCGCTCT6569              TATCGCGAAAAACGTAACGGTCCTCTCCTTTGTGAGAAATCAAATTACCGCGTAGTGAAA6629              CCGAAGCGCCATCGTGCATGGTTTTTGCGAAATCAACGGTCATTTTTTTTGCATCATCGG6689              TTCCGCGATAGCCATCTTCTATTGCATGAGGCGGCGGTGGCGCTGCATCCTGTTTTAAAC6749              CGCCCTGGTCATCTGCCAACGCATAAGGCATGACAAGAAAACTTGCTAATACAATGGCCT6809              GAAATTTCATACTAACTCCTTAATTGCGTTTGGTTTGACTTATTAAGTCTGGTTGCTATT6869              TTTATAATTGCCAAATAAGAATATTGCCAATTGTTATAAGGCATTTAAAATCAGCCAACT6929              AGCTGTCAAATATACAGAGAATTTAACTCACTAAAGTTAAGAAGATTGAAAAGTCTTAAA6989              CATATTTTCAGAATAATCGGATTTATATGTTTGAAAATTATTATATTGGACGAGCATACA7049              GAAAAAGCAAATCACCTTTACATATAAAAGCGTGGACAAAAAACAGTGAACATTAATAGA7109              GATAAAATTGTACAACTTGTAGATACCGATACTATTGAAAACCTGACATCCGCGTTGAGT7169              CAAAGACTTATCGCGGATCAATTACGCTTAACTACCGCCGAATCATGCACCGGCGGTAAG7229              TTGGCTAGCGCCCTGTGTGCAGCTGAAGATACACCCAAATTTTACGGTGCAGGCTTTGTT7289              ACTTTCACCGATCAGGCAAAGATGAAAATCCTCAGCGTAAGCCAGCAATCTCTTGAACGA7349              TATTCTGCGGTGAGTGAGAAAGTGGCAGCAGAAATGGCAACCGGTGCCATAGAGCGTGCG7409              GATGCTGATGTCAGTATTGCCATTACCGGCTACGGCGGACCGGAGGGCGGTGAAGATGGT7469              ACGCCAGCGGGTACCGTCTGGTTTGCGTGGCATATTAAAGGCCAGAACTACACTGCGGTT7529              ATGCATTTTGCTGGCGACTGCGAAACGGTATTAGCTTTAGCGGTGAGGTTTGCCCTCGCC7589              CAGCTGCTGCAATTACTGCTATAACCAGGCTGGCCTGGCGATATCTCAGGCCAGCCATTG7649              GTGGTGTTTATATGTTCAAGCCACGATGTTGCAGCATCGGCATAATCTTAGGTGCCTTAC7709              CGCGCCATTGTCGATACAGGCGTTCCAGATCTTCGCTGTTACCTCTGGAAAGGATCGCCT7769              CGCGAAAACGCAGCCCATTTTCACGCGTTAATCGCCCTGCTCAACAAACCACTGATAACC7829              ATCATCGGCCAACATTTGCGTCCACAGATAAGCGTAATAACCTGCAG7876                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 157 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetMetAspThrPheGlyIleSerIleProGlyLeuArgIleAlaGly                              151015                                                                        GlyLeuIleValAlaPheIleGlyPheArgMetLeuPheProGlnGln                              202530                                                                        LysAlaIleAspSerProGluAlaLysSerLysSerGluGluLeuGlu                              354045                                                                        AspGluProSerAlaAsnIleAlaPheValProLeuAlaMetProSer                              505560                                                                        ThrAlaGlyProGlyThrIleAlaMetIleIleSerSerAlaSerThr                              65707580                                                                      ValArgGlnSerSerThrPheAlaAspTrpValLeuMetValAlaPro                              859095                                                                        ProLeuIlePhePheLeuValAlaValIleLeuTrpGlySerLeuArg                              100105110                                                                     SerSerGlyAlaIleMetArgLeuValGlyLysGlyGlyIleGluAla                              115120125                                                                     IleSerArgLeuMetGlyPheLeuLeuValCysMetGlyValGlnPhe                              130135140                                                                     IleIleAsnGlyIleLeuGluIleIleLysThrTyrHis                                       145150155                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetLeuAspLeuPheLysAlaIleGlyLeuGlyLeuValValLeuLeu                              151015                                                                        ProLeuAlaAsnProLeuThrThrValAlaLeuPheLeuGlyLeuAla                              202530                                                                        GlyAsnMetAsnSerAlaGluArgAsnArgGlnSerLeuMetAlaSer                              354045                                                                        ValTyrValPheAlaIleMetMetValAlaTyrTyrAlaGlyAsnTrp                              505560                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 144 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysSerThrSerAspLeuPheAsnGluIleIleProLeuGlyArg                              151015                                                                        LeuIleHisMetValAsnGlnLysLysAspArgLeuLeuAsnGluTyr                              202530                                                                        LeuSerProLeuAspIleThrAlaAlaGlnPheLysValLeuCysSer                              354045                                                                        IleArgCysAlaAlaCysIleThrProValGluLeuLysLysValLeu                              505560                                                                        SerValAspLeuGlyAlaLeuThrArgMetLeuAspArgLeuValCys                              65707580                                                                      LysGlyTrpValGluArgLeuProAsnProAsnAspLysArgGlyVal                              859095                                                                        LeuValLysLeuThrThrGlyGlyAlaAlaIleCysGluGlnCysHis                              100105110                                                                     GlnLeuValGlyGlnAspLeuHisGlnGluLeuThrLysAsnLeuThr                              115120125                                                                     AlaAspGluValAlaThrLeuGluTyrLeuLeuLysLysValLeuPro                              130135140                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetThrMetSerArgArgAsnThrAspAlaIleThrIleHisSerIle                              151015                                                                        LeuAspTrpIleGluAspAsnLeuGluSerProLeuSerLeuGluLys                              202530                                                                        ValSerGluArgSerGlyTyrSerLysTrpHisLeuGlnArgMetPhe                              354045                                                                        LysLysGluThrGlyHisSerLeuGlyGlnTyrIleArgSerArgLys                              505560                                                                        MetThrGluIleAlaGlnLysLeuLysGluSerAsnGluProIleLeu                              65707580                                                                      TyrLeuAlaGluArgTyrGlyPheGluSerGlnGlnThrLeuThrArg                              859095                                                                        ThrPheLysAsnTyrPheAspValProProHisLysTyrArgMetThr                              100105110                                                                     AsnMetGlnGlyGluSerArgPheLeuHisProLeuAsnHisTyrAsn                              115120125                                                                     Ser                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetLysProLeuSerSerAlaIleAlaAlaAlaLeuIleLeuPheSer                              151015                                                                        AlaGlnGlyValAlaGluGlnThrThrGlnProValValThrSerCys                              202530                                                                        AlaAsnValValValValProProSerGlnGluHisProProPheAsp                              354045                                                                        LeuAsnHisMetGlyThrGlySerAspLysSerAspAlaLeuGlyVal                              505560                                                                        ProTyrTyrAsnGlnHisAlaMet                                                      6570                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 266 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetLeuAlaGlyLeuArgPheMetLeuValAlaPheProAlaIlePhe                              151015                                                                        PheValAlaArgProLysValProLeuAsnLeuLeuLeuGlyTyrGly                              202530                                                                        LeuThrIleSerPheAlaGlnPheAlaPheLeuPheCysAlaIleAsn                              354045                                                                        PheGlyMetProAlaGlyLeuAlaSerLeuValLeuGlnAlaGlnAla                              505560                                                                        PhePheThrIleMetLeuGlyAlaPheThrPheGlyGluArgLeuHis                              65707580                                                                      GlyLysGlnLeuAlaGlyIleAlaLeuAlaIlePheGlyValLeuVal                              859095                                                                        LeuIleGluAspSerLeuAsnGlyGlnHisValAlaMetLeuGlyPhe                              100105110                                                                     MetLeuThrLeuAlaAlaAlaPheSerTrpAlaCysGlyAsnIlePhe                              115120125                                                                     AsnLysLysIleMetSerHisSerThrArgProAlaValMetSerLeu                              130135140                                                                     ValIleTrpSerAlaLeuIleProIleIleProPhePheValAlaSer                              145150155160                                                                  LeuIleLeuAspGlySerAlaThrMetIleHisSerLeuValThrIle                              165170175                                                                     AspMetThrThrIleLeuSerLeuMetTyrLeuAlaPheValAlaThr                              180185190                                                                     IleValGlyTyrGlyIleTrpGlyThrLeuLeuGlyArgTyrGluThr                              195200205                                                                     TrpArgValAlaProLeuSerLeuLeuValProValValGlyLeuAla                              210215220                                                                     SerAlaAlaLeuLeuLeuAspGluArgLeuThrGlyLeuGlnPheLeu                              225230235240                                                                  GlyAlaValLeuIleMetThrGlyLeuTyrIleAsnValPheGlyLeu                              245250255                                                                     ArgTrpArgLysAlaValLysValGlySer                                                260265                                                                        __________________________________________________________________________

I claim:
 1. An isolated nucleic acid molecule comprising a moleculeselected from the group consisting of(a) a molecule having the sequenceof nucleotides 1893-2279 of SEQ ID NO:1, (b) a molecule which hybridizesunder stringent conditions to a molecule consisting of the sequence ofnucleotides 1893-2279 of SEQ ID NO:1, and (c) complements of (a) or (b),wherein the isolated nucleic acid molecule encodes an activator of abacterial multiple antibiotic resistance operon.
 2. The isolated nucleicacid of claim 1, wherein the isolated nucleic acid comprises SEQ IDNO:1.
 3. The isolated nucleic acid molecule of claim 1, wherein theisolated nucleic acid is isolated from a member of theEnterobacteriaceae.
 4. The isolated nucleic acid molecule of claim 1,wherein the isolated nucleic acid is free of operable sequences encodinga repressor locus of the bacterial multiple antibiotic resistanceoperon.
 5. The isolated nucleic acid molecule of claim 4 operably linkedto a regulatory region.
 6. The isolated nucleic acid molecule of claim 5in a plasmid, and wherein replication of the plasmid is temperaturesensitive.
 7. The isolated nucleic acid molecule of claim 1 wherein theisolated nucleic acid molecule is labeled.
 8. An isolated nucleic acidmolecule comprising a nucleic acid molecule having the nucleotidesequence of at least 15 contiguous nucleotides of the nucleic acid ofclaim
 1. 9. The isolated nucleic acid molecule of claim 8, wherein theisolated nucleic acid has the nucleotide sequence of at least 15contiguous nucleotides of nucleotides 1893-2297 of SEQ ID NO:1.
 10. Theisolated nucleic acid molecule of claim 8, wherein the isolated nucleicacid is free of operable sequences encoding a repressor locus of thebacterial multiple antibiotic resistance operon.
 11. The isolatednucleic acid molecule of claim 10 operably linked to a regulatoryregion.
 12. The isolated nucleic acid molecule of claim 11 in a plasmid,and wherein replication of the plasmid is temperature sensitive.
 13. Theisolated nucleic acid molecule of claim 8 wherein the isolated nucleicacid molecule is labeled.
 14. An isolated antisense nucleic acid whichhybridizes under physiological conditions to a MAR operon ortranscription product thereof, and which reduces expression of marA. 15.The isolated antisense nucleic acid of claim 14, comprising at least 15contiguous nucleotides of the isolated nucleic acid of claim
 1. 16. Theisolated antisense nucleic acid of claim 14, comprising at least 15contiguous nucleotides of SEQ ID NO:1 or the complement thereof.
 17. Theisolated antisense nucleic acid of claim 16, wherein the at least 15contiguous nucleotides are contained within nucleotides 1893-2279 of SEQID NO:1.
 18. The isolated antisense nucleic acid of claim 14, whereinthe isolated antisense nucleic acid is free of operable sequencesencoding a repressor locus of the bacterial multiple antibioticresistance operon.
 19. The isolated nucleic acid molecule of claim 17operably linked to a regulatory region.
 20. The isolated nucleic acidmolecule of claim 18 in a plasmid, and wherein replication of theplasmid is temperature sensitive.
 21. An antibacterial compositioncomprisingan antibiotic composition and a substance which binds to abacterial multiple antibiotic resistance operon, or a transcription orexpression product thereof, and which decreases the expression of marA.22. The antibacterial composition of claim 21 wherein the substance isan isolated antisense nucleic acid molecule which hybridizes underphysiological conditions to the multiple antibiotic resistance operon ora transcription product thereof.
 23. The antibacterial composition ofclaim 22, wherein the antisense nucleic acid molecule comprises at least15 contiguous nucleotides of SEQ ID NO:1 or the complement thereof. 24.The antibacterial composition of claim 22, wherein the antisense nucleicacid molecule comprises at least 15 contiguous nucleotides of theisolated nucleic acid of claim
 1. 25. A bacterial cell into which hasbeen introduced an isolated nucleic acid molecule selected from thegroup consisting of the isolated nucleic acid molecule of claim 5, theisolated nucleic acid molecule of claim 6, the isolated nucleic acidmolecule of claim 11, the isolated nucleic acid molecule of claim 12,the isolated nucleic acid molecule of claim 17 and the isolated nucleicacid molecule of claim
 18. 26. The bacterial cell of claim 22, whereinthe bacteria are recombination deficient.
 27. The bacterial cell ofclaim 22 or claim 23, wherein the bacteria are free of an operablebacterial multiple antibiotic resistance operon.